The interaction of Insulin-like Growth Factor--1 (IGF-1) with its own receptor (IGF-1 R) seems to play a major role in normal development and in the control of both normal and abnormal growth. In growth hormone disturbances of growth as, for instance, in acromegalics and in patients with growth hormone deficiency, clinical assessments of disease activity correlate far better with blood levels of IGF-1 than they do with growth hormone concentrations, Van Wyk et al., The Biology of Normal Human Growth, pp. 223-239, Raven Press, N.Y. (1981). Werner et al., Proc. Nat. Acad. Sci. USA, 86:7451-5 (1989) have shown that the mRNA levels for the IGF-1 R decrease steadily in all tissues during post-natal development, reaching a maximum during the perinatal stages. IGF-1 mRNA, instead, is not so tightly regulated during development as the mRNA for the IGF-1 R, and actually reaches maximum expression in the adult liver, which is the main site of production of IGF-1. Apart from these general considerations, a number of reports have appeared indicating that the interaction of IGF-1 with its own receptor may play a major role in cell growth. For instance, IGF-1 receptors are present in phytohemagglutinin activated T lymphocytes, Kozak et al., Cell Immunol , 1009 318-331 (1987) and in K562 cells that are a human erythroleukemia cell line, Hizuka et al., Endocrinol. Japon, 34:81-88 (1987). In fact, K562 cells grow vigorously in SFM containing only IGF-1 or supraphysiological concentrations of insulin. An abundance of IGF-1 receptors has also been reported in lymphoblasts of human T cell leukemias, Lee et al., J. Clin. Endocrinol. & Metabol., 62:28-35 (1986), and in HL60 cells, Pepe et al., J. Cell Physiol., 133:219-227 (1987). In our own laboratory, we have been able to show that the mRNA for the IGF-1 receptor is over-expressed in HL60 cells. Again, HL60 cells, as well as other cell lines, grow well in serum-free medium containing only insulin in supraphysiological concentrations. In Burkitt cells, the number of IGF-1 receptors increase between G.sub.1 and S-3 phase, Hartman et al., Leukemia, 2:241-4 (1988). Stem cells and progenitor cells also seem to require IGF-1 for growth. Goldring and Goldring, Eucar. Gene Express, 1:-301-326 (1991), list several references indicating that IGF-1 increases the proliferation of keratinocytes, smooth muscle cells, osteoblasts, chrondrocyts and neuronal cells (see their Table 4). The IGF-1 R is induced by estrogens in breast cancer cell lines, Stewart et al., J. Biol. Chem., 265:21172-8 (1990), Pekonen et al., Cancer Res., 48:1343-7 (1988), Peyrat et al., Cancer Res., 48:6429-33 (1988), Foekens et al., Cancer Res,, 49:5823-8 (1989), and the expression of IGF-1 receptors seems to correlate with the growth of breast cancer, at least just as well as the estrogen receptors or the EGF receptor. Other tumors in which an increased expression of IGF-1 R or, at least, IGF-1 binding sites, have been reported include small cell lung cancer, Kiefer et al., Exp. Cell Res., 184:396-406 (1989), Minuto et al., Cancer Res., 48:3716-9 (1988), Nakanishi et al., J. Clin. Invest., 82:354-9 (1988), choriocarcinoma cells, Ritvos et al., Endocrinology, 122:395-401 (1988), malignant glioma, Gammeltoft et al., Cancer Res., 48:1233-7 (1988), renal carcinoma, Pekonen et al., Int. J. Cancer, 43:1029-33 (1989), and neoplastic human endometrium, Talavera et al., J. Cancer Res., 50:3019-24 (1990). A role of the IGF-1 R in growth has also been reported in human melanoma cells, Stracke et al., J. Biol. Chem., 264:21544-9 (1989), and in tumors of neural origins like neuroblastomas or pheochromocytomas, Ota et al., Molec. Brain Res., 6:69-76 (1989) and Ota et al., Cur. J. Biochem., 174:521-30 (1988). However, the best evidence that the IGF-1 R plays a major role in the control of cellular proliferation comes from studies with fibroblasts in cell cultures.
It has been known for a long time that IGF-1 is necessary for the growth of fibroblasts in vitro. Prototypes for growth studies have been the 3T3 mouse cells and the WI38 human diploid fibroblasts. With BALB/c3T3 cells, at least two poor plasma (ppp), are both necessary for sustained growth, Scher et al., Biochem. Biophys. Acta, 560:217-41 (1979). PPP can be replaced by IGF-1, or by insulin at high concentrations, but IGF-1 only (without PDGF) does not stimulate the growth of 3T3 or WI-38 cells, Stiles et al., Proc. Natl. Acad. Sci. USA, 76:1279-83 (1979), Leof et al., Exp. Cell Res., 141:107-15 (1982), Russell et al., Proc. Natl. Acad. Sci. USA, 81:2389-92 (1984), Gai et al., Oncogene Res., 3:377-86 (1988). Other cell lines, as for instance BHK cells, Cherington et al., Proc. Natl. Acad.. Sci., 76:3937-41 (1979), and WI38 human diploid fibroblasts, Phillips et al., Exp. Cell Res., 175:396-403 (1988), also require more than one growth factor for optimal growth in culture, usually PDGF (or EGF) and IGF-1. PDGF and/or EGF can actually be replaced but, until now, IGF-1 has not been replaced. For instance, PDGF can be replaced by an overexpressed c-myc, Armelin et al., Nature (London), 310:655-60 (1984) and Kaczmarek et al., Science, 228:1313-5 (1985), or even, under certain circumstances, by exposure to cycloheximide, Kaczmarek et al., Cell Biol. Int. Rept., 10:455-63 (1986), and in WI38 EGF and/or PDGF can be replaced by high concentrations of calcium, Praeger et al., In Vitro, 22:355-9 (1986). Travali et al., Mol. Cell Biol., 11:731-736 (1991), disclose that IGF-1 can be replaced by a constitutively expressed c-myb. However, it was found that in cells constitutively expressing c-myb, there was a marked increase in IGF-1 mRNA and IGF-1 secretion so that at least in this instance, although myb can replace IGF-1, it does so by simply stimulating the production of IGF-1.
The fact that these cells respond to IGF-1 plus PDGF may make 3T3 or WI38 cells sensitive to IGF-1 simply by increasing the number of IGF-1 binding sites. It has been shown that the addition of PDGF and/or EGF to BALB/c3T3 cells or to WI38 human diploid fibroblasts, increases both the production of IGF-1 and the number of IGF-1 binding sites. However, until the present invention, it was not known that the constitutive expression of IGF-1 and IGF-1 R could abrogate all requirements for exogenous growth factors.